Nitrogenases carry out the conversion of N2 to ammonium, a form of N that is more readily available for human nutrition. At the active site of nif-encoded, molybdenum nitrogenase is the iron-molybdenum cofactor, FeMo-co. The vnf-encoded nitrogenase contains the structurally similar iron-vanadium cofactor, FeV-co. The overall goal of this project is to determine the pathways of biosynthesis of FeMo-co and FeV-co in the nitrogen-fixing procaryote, Azotobacter vinelandii. The nifBUSV gene products are required for synthesis of both cofactors. In addition, the nifNE, nifH and nifX products are required for the synthesis of FeMo-co. When cells are grown on V in place of Mo, the cell synthesizes vnfNE, vnfH and vnfX gene products for the synthesis of FeV-co. The specific goals of this project are to define the roles of these gene products in the syntheses of FeMo-co/FeV-co and to establish the basis for the specificity for Mo or V during synthesis of each cofactor. NifB-co, the metabolic product of NifB, is an Fe and S donor to FeMo-co and FeV-co; the determination of the structure of NifB-co is a goal of this project. Several proteins in the FeMo-co synthesis pathway including NifH accumulate label from 55Fe-NifB-co or from 99MoO42- during the in vitro synthesis of FeMo-co, and 55Fe and 99Mo will be used to follow the steps in the pathway. Likewise, 49V accumulates on several proteins during FeV-co synthesis, and 55Fe and 49V will be used to follow the steps of the FeV-co pathway. VnfX has recently been identified as a protein that accumulates V and Fe during FeV-co synthesis, and the investigation of the role of VnfX in FeV-co synthesis will be a major goal of the project. All organisms require trace elements, and the information gained in this project will increase our understanding of the mechanisms by which living cells assimilate, accumulate, and discriminate between trace elements that are used to build complex cofactors required for many redox reactions crucial to the cell. Human health is dependent on proper metabolism and utilization of trace elements such as those being investigated in this study.